The present invention discloses a method to produce sterile male flowers and seedless or parthenocarpic fruits. This method is based on silencing genes that are homologous to Pistillata genes from different plant species, by generating silencing vectors that comprise the entire sequence of a Pistillata-homologous gene specific for the used species or a part of this sequence, and the use of said genetic silencing vectors to produce sterile male flowers and seedless or parthenocarpic fruits.
In the present invention, the sequences of VvPI gene from Vitis vinifera (grapevine) cv. Cabernet Sauvignon and LePI gene from Lycopersicon esculentum (tomato) are further disclosed, together with the use of these genes to produce sterile male flowers and seedless or parthenocarpic fruits. Furthermore, silencing vectors that comprise these sequences or a part thereof contained in said silencing vectors are disclosed, which allow silencing VvPI and LePI genes in Vitis vinifera cv. Cabernet Sauvignon and Lycopersicon esculentum, respectively.
VvPI from Vitis vinifera cv. Cabernet Sauvignon (grapevine) and LePI from Lycopersicon esculentum (tomato) are genes that are homologous to the Pistillata gene from Arabidopsis thaliana, which is a gene involved in petal and stamen formation in Arabidopsis and other flowers.
Seedless varieties have a high commercial value and improved organoleptic properties. Moreover, seedless varieties solve problems in some cultures, such as pollination. In the production of various cultivars, such as Lycopersicon esculentum, pollination use to be a critical step and even a limiting step for production, since this step is affected by wind, humidity and temperature, both in sub- or supra-optimal conditions (Nothman et al., 1975; Romano et al., 1994). Parthenocarpy offers an alternative route, because fruit development can occur independently from pollination (Lukyanenko et al., 1991) and thus it assures a greater stability to the producer.
In the majority of flowering plants, once pollination and subsequent fertilization have taken place, ovules develop to seeds, while the surrounding tissue differentiates to the fruit (Coombe et al., 1975). Fertilization is a critical step to begin flower-to-fruit transition, except for those parthenocarpic plants in which fruit development is uncoupled to fertilization. Parthenocarpic fruits develop without previous fertilization of the ovules, thus these ovules grow senescent and the ovary develops to a seedless fruit (Gillaspy et al., 1993).
Two are the phenomena associated with the apparition of seedless (apyrenic) fruits: parthenocarpy and stenospermocarpy. While parthenocarpy is the development and growth of a fruit without previous fertilization, stenospermocarpy is the apparition of seedless fruits or a reduced number of partially-formed seeds once fertilization has taken place, which is caused by an abortion of the seed during its formation.
The first methodology used to artificially create apyrenic fruits consisted of spraying developing fruits and even flowers, with auxins and gibberellins (GAs) (Nitsch et al., 1970; Schwabe et al., 1981; García-Martínez et al., 1997). However, the most used methodology to produce seedless fruits has been mutant selection. Many of these seedless mutant variants have been obtained by induced mutagenesis. For example, in tomato (Lycopersicon esculentum) different parthenocarpic lines have been produced in this way (reviewed by Lukyanenko et al., 1991). The most important cultivar line is the mutant obtained by using ethyl methane-sulfonate (Bianchi et al., 1969), called pat (parthenocarpic fruit) (Soressi and Salamini, 1975; Philouze et al., 1983; Barg et al., 1990; George et al., 1984; Lukyanenko et al., 1991).
Polyploidy has also been used, such as in triploid watermelon (Terada et al., 1943; Kihara et al., 1951, 1958) and citrus (Deng et al., 1996; Chandler et al., 2000; Guo et al. 2000) mutants. These mutants are generated by fusing protoplasts or crossing a tetraploid (4×) maternal plant with a diploid (2×) pollinator, thus generating a triploid (3×) individual that is then pollinated by a diploid plant to obtain seedless fruits. A more novel way comprises pollinating flowers with irradiated pollen. As was shown by Watanabe (2001) and Sugiyama (2002), this method allows obtaining diploid (2×) watermelon plants that produce fruits having few soft and small seeds, known as “empty seeds”.
Lately, parthenocarpic fruits have been obtained using an approximation analogous to exogenous hormone application. The procedure comprises altering the phytohormone production pathway by introducing genes as iaaM, iaaH and rolB that increase internal hormone levels in the ovary, ovules and placenta. Szechtman et al. (1997) expressed gene iaaH from Agrobacterium tumefaciens specifically in tomato ovary. This gene codes for an indoleacetamide hydrolase that hydrolyzes naphthaleneacetamide (NAM) to naphthaleneacetic acid (NAA), an active auxin form. Thus, parthenocarpy was induced upon subsequently treating the ovary with NAM. Rotino et al. (1996) expressed a gene that codes for an enzyme involved in indol-3-acetic acid (IAA) biosynthesis that does not need any exogenous application of compounds. Said gene was the chimerical DefH9-iaaM gene, which has the codifying region of iaaM gene from Pseudomonas syringae pv. savastanoi and the placenta and ovule-specific promoter region DefH9 from Antirrhinum majus (Rotino et al., 1996). Gene iaaM codes for an enzyme, tryptophan monooxygenase, that produces indoleacetamide. Indoleacetamide is then enzymatically or chemically converted into the IAA auxin. Tobacco (Rotino et al., 1997), eggplant (Donzella et al., 2000; Rotino et al., 1997; Acciarri et al., 2002), Lycopersicon esculentum (Ficcadenti et al., 1999; Pandolfini et al., 2002) and strawberry and raspberry (Mezzetti et al., 2002; 2004) transgenics that have the transgene DefH9-iaaM are parthenocarpic in nature.
It has been previously shown that parthenocarpic apple (Malus domestica) varieties: Rae Ime, Spencer Seedless and Wellington Bloomless, had one transposon-insertion mutation in the orthologous Pistillata (PI) gene: MdPI (Malus domestica Pistillata). Furthermore, this PI gene requires the expression of Sepallata to carry out its function in Arabidopsis (Honma and Goto 2000; Pelaz et al. 2000, 2001). Therefore, lack of expression of genes that are homologous to tomato Sepallata (such as TM29, Ampomah-Dwamena et al. 2002; and TM5, Pnueli et al. 1994), leads to parthenocarpic fruit development.
Genetic Silencing.
Genetic silencing is a natural mechanism that allows gene expression inhibition at DNA level (transcriptional genetic silencing) or at messenger RNA level (post-transcriptional genetic silencing, PTGS). Currently, procedures have been developed to allow inhibiting gene expression by PTGS in various organisms, including plants. To this end, silencing plasmid vectors are used (e.g., Hellsgate) that generate sense and antisense RNA strands, thus forming double strand RNAs in the cytoplasm, which induce silencing of the target gene that has a similar sequence to that of the double RNA (Voinet, 2003).
In this invention, this technology was used to induce silencing of Pistillata genes from Vitis vinifera and Lycopersicon esculentum (grapevine and tomato, respectively), which are genes codifying for transcriptional factors responsible for petal and stamen formation in flowers.
Among the closest documents to this invention, the following could be mentioned:
Vliet G., 1998 (Patent Application WO 98/24301): this document claims a method to produce seedless tomatoes by crossing two homozygote tomato plants that are recessive for parthenocarpy (pk,pk) and functional sterility (fs,fs) characteristics; this is a technique totally different to that disclosed in the present invention.
In Ito et al., 2002 (US Patent Application US 2002/0152495), a polynucleotide is described that comprises a sequence codifying for cytochrome P450, and when this sequence is expressed in a plant, a parthenocarpic phenotype is produced and furthermore a larger fruit is obtained; this technique also differs from that of the present invention in the type of gene used and the mechanism by which it operates.